Lipids that have a sphingoid backbone as a common structure such as a sphingoglycolipid, a sphingophospholipid (including a sphingophosphonolipid) and a ceramide are collectively designated as sphingolipids. The amino group of the sphingoid backbone binds a long chain fatty acid having heterogeneous chain length through an acid amide bond to form a ceramide.
Recently, it has proved that the sphingolipid is widely distributed in organisms including lower animals and higher animals and plays important roles in biological activities such as growth, induction of differentiation and apoptosis in cells. In addition, the sphingolipid, which is a component of cellular membrane, has been used as an additive for cosmetics or pharmaceuticals.
An N-deacylated form (lyso-form) of the sphingolipid that lacks the fatty acid, which is attached to the amino group of the sphingoid in the sphingolipid through the acid amide bond, is designated as a lysosphingolipid. It has proved that the lysosphingolipid has similar and different biological activities as compared with those of the sphingolipid.
Since the lysosphingolipid has a free amino group in the sphingoid, it is useful as a starting material for synthesizing a lysosphingolipid derivative (a sphingolipid derivative or a sphingolipid analogue) by re-acylation. For example, a sphingolipid having a homogeneous fatty acid composition, a sphingolipid having different fatty acid chain length from that of the starting sphingolipid, or a novel sphingolipid containing a functional fatty acid such as docosahexaenoic acid can be synthesized. Alternatively, a sphingolipid labeled with a chromophore, a radioisotope or the like can be obtained. Furthermore, immobilization to a carrier can be achieved by utilizing the free amino group of the lysosphingolipid.
Conventional processes for producing a lysosphingolipid include a chemical process, a process in which an enzyme is used and a process in which a microorganism is used.
A hydrazinolysis method and a method of alkaline hydrolysis in an alcoholic solvent are known for chemically obtaining a lysosphingolipid from a sphingoglycolipid. However, when a glycosphingolipid that contains a sialic acid (a ganglioside) is used, for example, deacylation reaction of the sialic acid portion proceeds simultaneously in such methods. Furthermore, the N-acetyl group is eliminated when a sphingoglycolipid that contains an amino sugar is used, resulting in a de-N-acetyl lysoglycolipid. A very complicated procedure is required to make the deacylated lysoglycosphingolipid to have a sugar chain identical with a naturally occurring one. In one exemplary procedure, a protective group is selectively introduced to the amino group of the lipid portion, the sialic acid portion is re-acylated, and the protective group is then detached. In another exemplary procedure, the sugar portion is selectively re-acylated after incorporation into liposome. Furthermore, various by-products are generated in these procedures. As described above, a great deal of labor and a technical skill are required for producing a lysosphingolipid by a chemical process.
A process using hydrochloric acid-hydrolysis in an alcoholic solvent is generally used for chemically obtaining a lyso-form of one of sphingophospholipids, a sphingomyelin. However, various by-products are generated by using such a process. For example, a steroisomer with a sphingoid of L-threo-type (2S, 3S) is formed in addition to a naturally occurring one of D-erythro-type (2S, 3R). Consequently, the yield of the naturally occurring D-erythro-form of interest is low, and it is very difficult to purify the D-erythro-form from the reaction mixture.
On the other hand, processes in which an enzyme that generates a lyso-form from a sphingolipid is used are known to date. The following processes are known: a process in which ganglioside ceramidase produced by an actinomycete of genus Nocardia is used [Journal of Biochemistry, 103:1–4 (1988); JP-A 64-60379]; a process in which an enzyme produced by an actinomycete of genus Rhodococcus or a processed product of the cells is used [JP-A 6-78782]; a process in which sphingolipid ceramide N-deacylase produced by a bacterium of genus Pseudomonas is used [Journal of Biological Chemistry, 270:24370–24374 (1995); JP-A 8-84587]; a process in which sphingolipid ceramide N-deacylase produced by a non-fermentative Gram-negative bacillus AI-2 is used [JP-A 10-257884]; and a process in which ceramidase produced by a bacterium of genus Pseudomonas s used [JP-A 10-14563].
However, the reaction yield of a lyso-form in the processes using these enzymes is not satisfactory. For example, when sphingolipid ceramide N-deacylase is used, although the yield varies depending on the substrate used, the yield from ganglioside GM2 (which is the most susceptible substrate to hydrolysis) is at most approximately 70%, whereas that from ganglioside GM1 is at most approximately 60%.
Processes for producing a lysosphingolipid in which the following microorganisms or extracts therefrom are used are known: an actinomycete of genus Streptomyces capable of producing glycosphingolipid ceramide deacylase [Bioscience, Biotechnology, and Biochemistry, 59:2028–2032 (1995); JP-A 7-107988]; and a bacterium of genus Pseudomonas or genus Shewanella which produces sphingolipid ceramide N-deacylase [JP-A 10-45792].
However, a reaction contains a large amount of impurities such as a dye, a glycolipid or the like derived from a culture medium or cells in addition to a lyso-form of interest in a process in which a substrate is added to the medium. Therefore, a complicated purification procedure has been required to obtain the lyso-form with high purity in such a process. Furthermore, it has been very difficult to handle a small amount of the sphingolipid to purify the lyso-form in the process for the reasons as described above.
In all of the conventional processes for producing a lysosphingolipid by using an enzyme, or a microorganism or an extract therefrom, the reaction is conducted only in an aqueous system. No process using a two-phase system consisting of an aqueous phase and a phase of an organic solvent that forms a separation phase immiscible with the aqueous phase has been known.